U.S. patent number 9,782,400 [Application Number 14/956,620] was granted by the patent office on 2017-10-10 for oncogenic ros1 and alk kinase inhibitor.
This patent grant is currently assigned to Macau University of Science and Technology. The grantee listed for this patent is Macau University of Science and Technology. Invention is credited to Lai Han Leung, Liang Liu, Lian Xiang Luo, Xiao Jun Yao.
United States Patent |
9,782,400 |
Yao , et al. |
October 10, 2017 |
Oncogenic ROS1 and ALK kinase inhibitor
Abstract
A compound suitable for treating cancer, in particular NSCLC,
inhibits activity of oncogenic ROS1 kinase and ALK kinase. The
compound has certain structural components such as a quinoline
moiety in the backbone and at least one phenyl-containing moiety in
a side chain with a hydrophobic substituent attached to the
backbone via an up to 6-membered linking group as well as a further
hydrophobic moiety. The presence of the structural components
allows for an advantageous interaction with the ROS1 kinase domain
and, further, with the ALK kinase domain. Hence, said compound
represents a highly promising opportunity for patients bearing
ROS1- or ALK-dependent cancer. A composition, in particular a
pharmaceutical composition, includes the compound. A method for
targeting cancer cells harboring an abnormality in ROS1 gene or ALK
gene includes contacting a cell with the compound.
Inventors: |
Yao; Xiao Jun (Taipa,
CN), Leung; Lai Han (Taipa, CN), Luo; Lian
Xiang (Taipa, CN), Liu; Liang (Taipa,
CN) |
Applicant: |
Name |
City |
State |
Country |
Type |
Macau University of Science and Technology |
Taipa, Macau |
N/A |
CN |
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Assignee: |
Macau University of Science and
Technology (MO)
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Family
ID: |
57586833 |
Appl.
No.: |
14/956,620 |
Filed: |
December 2, 2015 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20160367547 A1 |
Dec 22, 2016 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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14744042 |
Jun 19, 2015 |
9526722 |
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Foreign Application Priority Data
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Jun 24, 2015 [AU] |
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2015100840 |
Nov 26, 2015 [AU] |
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2015101722 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K
31/4745 (20130101) |
Current International
Class: |
A01N
43/42 (20060101); A61K 31/4745 (20060101); A61K
31/444 (20060101) |
Other References
Lim et al, Plos One, Sep. 14, 2015. cited by examiner .
Mazieres et al. JCO, Feb. 2015. cited by examiner .
Rolfo et al, Transl Lung Cancer Res. Aug. 2014; 3(4): 250-261.
cited by examiner .
Freshney (Culture of Animal Cells, A Manual of Basic Technique,
Alan R. Liss, Inc., 1983, New York, p. 4). cited by examiner .
Dermer (Bio/Technology, 1994, 12:320). cited by examiner .
Gura (Science, v278, 1997, pp. 1041-1042). cited by examiner .
Jemal, A. et al. Cancer statistics, 2009. CA: a cancer journal for
clinicians 59, 225-249 (2009). cited by applicant .
Shaw, A.T., Hsu, P.P., Awad, M.M. & Engelman, J.A. Tyrosine
kinase gene rearrangements in epithelial malignancies. Nature
reviews. Cancer13, 772-787 (2013). cited by applicant .
Looyenga, B.D., Cherni, I., Mackeigan, J.P. & Weiss, G.J.
Tailoring tyrosine kinase inhibitors to fit the lung cancer genome.
Translational oncology4, 59-70 (2011). cited by applicant .
Christine M. Lovly1 and William Pao1 Escaping ALK inhibition
mechanisms of and strategies to overcome resistance. Science
translational medicine4, 1-5 (2012). cited by applicant .
Takeuchi, K. et al. RET, ROS1 and ALK fusions in lung cancer.
Nature medicine18, 378-381 (2012). cited by applicant .
Kwak, E.L. et al. Anaplastic lymphoma kinase inhibition in
non-small-cell lung cancer. The New England journal of medicine363,
1693-1703 (2010). cited by applicant .
Roskoski, R., Jr. Anaplastic lymphoma kinase (ALK): structure,
oncogenic activation, and pharmacological inhibition.
Pharmacological research68, 68-94 (2013). cited by applicant .
Gandhi, L. & Janne, P.A. Crizotinib for ALK-rearranged
non-small cell lung cancer: a new targeted therapy for a new
target. Clinical cancer research : an official journal of the
American Association for Cancer Research18, 3737-3742 (2012). cited
by applicant .
Alamgeer, M., Ganju, V. & Watkins, D.N. Novel therapeutic
targets in non-small cell lung cancer. Current opinion in
pharmacology13, 394-401 (2013). cited by applicant .
Hallberg, B. & Palmer, R.H. Mechanistic insight into ALK
receptor tyrosine kinase in human cancer biology. Nature reviews.
Cancer13, 685-700 (2013). cited by applicant .
Katayama, R. et al. Mechanisms of acquired crizotinib resistance in
ALK-rearranged lung Cancers. Science translational medicine4,
120ra117 (2012). cited by applicant .
Zou, H.Y. et al. PF-06463922 is a potent and selective
next-generation ROS1/ALK inhibitor capable of blocking
crizotinib-resistant ROS1 mutations. Proceedings of the National
Academy of Sciences of the United States of America112, 3493-3498
(2015). cited by applicant .
Ou, S.H., Tan, J., Yen, Y. & Soo, R.A. ROS1 as a `druggable`
receptor tyrosine kinase: lessons learned from inhibiting the ALK
pathway. Expert review of anticancer therapy12, 447-456 (2012).
cited by applicant.
|
Primary Examiner: Cornet; Jean
Attorney, Agent or Firm: Renner Kenner Greive Bobak Taylor
& Weber
Claims
The invention claimed is:
1. A method of treating a subject suffering from non-small cell
lung cancer comprising administering an effective amount of a
compound of Formula (Ic) or a pharmaceutically acceptable salt,
solvate or anhydrate thereof to the subject: ##STR00015##
2. A method of Inhibiting ROS1 kinase activity or ALK kinase
activity in non-small cell lung cancer cells comprising
administering an effective amount of a compound of Formula (Ic) or
a pharmaceutically acceptable salt, solvate or anhydrate thereof to
a subject suffering from cancer: ##STR00016##
3. The method of claim 1, wherein the subject is a mammal having an
abnormality in ROS1 gene resulting from a ROS1 chromosome
rearrangement.
4. The method of claim 3, wherein the ROS chromosome rearrangement
is associated with the expression of at least one of SLC34A2-ROS1
or CD74-ROS1 fusion kinase.
5. The method of claim 1, wherein the subject is a mammal having an
abnormality in ALK gene resulting from an ALK chromosome
rearrangement.
6. The method of claim 5, wherein the ALK chromosome rearrangement
is associated with the expression of at least one EML4-ALK fusion
kinase.
7. A method for targeting non-small cell lung cancer cells
harboring an abnormality in ROS1 gene or an abnormality in ALK gene
comprising the step of contacting said cells with a compound of
Formula (Ic) or a salt, solvate or anhydrate thereof:
##STR00017##
8. The method of claim 7, wherein the proliferation of the
non-small cell lung cancer cells is Inhibited, reduced or prevented
or apoptosis of the non-small cell lung cancer cells is
Induced.
9. The method of claim 7, wherein the non-small cell lung cancer
cells are from a lung tumor.
10. The method of claim 7, wherein the non-small cell lung cancer
cells are from NSCLC adenocarcinoma.
11. The method of claim 7, wherein the non-small cell lung cancer
cells harbor an abnormality in ROS1 gene, and wherein said
abnormality is a ROS1 chromosome rearrangement associated with the
expression of at least one of SLC34A2-ROS1 or CD74-ROS1 fusion
kinase.
12. The method of claim 11, wherein the compound has an IC.sub.50
on the non-small cell lung cancer cells of at most 10 .mu.M and an
IC.sub.50 on non-cancerous lung cells being at least 2.5 times
higher than the IC.sub.50 on the non-small cell lung cancer
cells.
13. The method of claim 7, wherein the non-small cell lung cancer
cells harbor an abnormality in ALK gene, and wherein said
abnormality is an ALK chromosome rearrangement and wherein the ALK
chromosome rearrangement Is associated with the expression of at
least one EML4-ALK fusion kinase.
14. The method of claim 13, wherein the compound has an IC.sub.50
on the non-small cell lung cancer cells of at most 10 .mu.M and an
IC.sub.50 on normal non-cancerous lung cells being at least 2 times
higher than the IC.sub.50 on the non-small cell lung cancer
cells.
15. The method of claim 7, wherein the compound of Formula (Ic) is
used in a concentration of at least 1.25 .mu.M.
16. The method of claim 7, wherein the concentration of the
compound of Formula (Ic) is at least 2.5 .mu.M.
17. The method of claim 7, wherein the non-small cell lung cancer
cells are contacted with the compound for at least 12 h.
Description
TECHNICAL FIELD
The present invention relates to a compound that can, in
particular, inhibit ROS1 kinase activity and ALK kinase activity
for treating cancer such as lung cancer like ROS1-dependent
non-small cell lung cancer or ALK-dependent non-small cell lung
cancer as well as compositions such as pharmaceutical compositions
comprising said compound. The present invention further provides a
method to target cancer cells harboring an abnormality in ROS1 gene
or ALK gene.
BACKGROUND OF INVENTION
Lung cancer is the leading cause of cancer-related mortality in
China and the world, wherein non-small cell lung cancer (NSCLC), in
particular NSCLC adenocarcinoma, accounts for approximately 85% of
all cases (Jemal, A. et al., CA: a cancer journal for clinicians,
2009, 59:225-249). There are more than 90 kinds of tyrosine kinases
which are related to NSCLC.
Receptor tyrosine kinases (RTKs) are mediators of extracellular
signals through activation of downstream signaling pathways
including ERK, AKT and/or STAT3 cascades to control cell growth,
proliferation, survival and motility pathways. In particular,
chromosome rearrangements, gene amplification, and point mutations
in respective genes contribute to and/or result in abnormal and
constitutive RTK activation which is in turn responsible for
initiation and progression of many cancers, including NSCLC. The
first targetable RTK identified in NSCLC was the anaplastic
lymphoma kinase gene (ALK), wherein chromosomal rearrangements of
ALK have been identified amongst which is as most common form the
echinoderm microtubule-associated protein-like 4 (EML4)-ALK, i.e.
comprising portions of the EML4 gene and the ALK gene, wherein
several variants of EML4-ALK gene fusions have been identified.
EML4-ALK gene fusions have been found in 3% to 7% of NSCLC
(Takeuchi, K. et al., Nature medicine, 2012, 18:378-381, Kwak, E.
L. et al., The New England journal of medicine, 2010,
363:1693-1703, Roskoski, R., Jr., Pharmacological research, 2013,
68:68-94). These percentages translate into significant numbers of
patients due to the increasing number of NSCLC (Shaw, A. T. et al.,
Nature, 2013, 13:772-787). Furthermore, additional fusion partners
besides EML4 have been identified and, besides, ALK activating
point mutations and presence of additional gene copies have been
observed in several further cancer types activating the signaling
pathways downstream to ALK (Roskoski, R., Jr., Pharmacological
research, 2013, 68:68-94). In the majority of cases, ALK chromosome
rearrangements are non-overlapping with other gene abnormalities
found in NSCLC, i.e. usually abnormalities in ALK and ROS1 gene
each define a distinct patient subgroup (Alamgeer, M. et al.,
Current opinion in pharmacology, 2013, 13:394-401).
Chromosome rearrangement involving the oncogenic c-ros oncogenel
(ROS1) RTK were later reported, wherein ROS1 gene has been found to
be fused with several gene partners in NSCLC. Approximately 1% to
2% of NSCLC patients harbor multiple kinds of ROS1 chromosome
rearrangement (Shaw, A. T. et al., Nature, 2013, 13:772-787).
Chromosome rearrangements of either ROS1 or ALK which may be based
on interchromosomal translocation or intrachromosomal deletion are
accompanied by the fusion of a portion of the ROS1 or ALK protein
that includes its entire tyrosine kinase domain with several
partner proteins with resulting ROS1 fusion kinases or ALK fusion
kinases being constitutively activated and driving cellular
transformation (e.g. Lovly, C. M. and Pao, W., Science
translational medicine, 2012, 4:1-5). Respective cancers become
dependent on continued signaling triggered by said fusion kinases,
also named "oncogene addiction" (Shaw, A. T. et al., Nature, 2013,
13:772-787). In particular, ROS1 or ALK fusion kinases activate
growth and survival pathways necessary for the growth and survival
of cancer cells, which pathways are reported to include
auto-phosphorylation of either ROS1 or ALK and phosphorylation of
AKT, ERK and STAT3.
Recent developments in targeted-based therapies have led to a major
paradigm shift in oncology. Small-molecule tyrosine kinase
inhibitors are provided to treat cancer patients who have tyrosine
kinase gene fusions, such as ROS1 or ALK chromosome rearrangements.
Several tyrosine kinase inhibitors proved to have promising effects
in the clinical practice. For example, crizotinib, a potent
ATP-competitive small molecule inhibitor of ALK, have now been
approved by the FDA for treating NSCLC patients that harbor ALK
rearrangements. Crizotinib shows marked anti-tumor activity both in
vitro and in vivo as well as in clinical practice. Since the
tyrosine kinase domains of ALK and ROS1 are very similar, with 77%
identity within the ATP-binding sites, most ALK inhibitors have
cross activity against ROS1. In one early clinical trial of
crizotinib to treat NSCLC patients harboring ROS1 rearrangements,
the objective response rate was 72%, the median duration of
response was 17.6 months and median progression-free survival was
19.2 months. Although most patients with ROS1-positive NSCLC
exhibit substantial clinical benefit from crizotinib, the efficacy
of crizotinib is limited due to the development of drug
resistances. Hence, ensuring durable response to crizotinib therapy
represents a universal challenge as drug resistance proved to be
common and based on several resistance mechanisms in patients
treated with crizotinib. Accordingly, patients who responded to
crizotinib will eventually experience disease progression despite
continued treatment.
Thus, further potent RTK inhibitors for cancer therapy have to be
identified. Accordingly, there is a strong need for new compounds
which are able to target RTKs and sufficiently inhibit their kinase
activity, in particular ROS1 or ALK kinase activity, which
compounds can, thus, be used for cancer therapy, in particular for
treatment of NSCLC.
SUMMARY OF INVENTION
The first aspect of the present invention relates to a method of
treating cancer, i.e. in particular lung cancer such as NSCLC, by a
compound of Formula (Ia) in a subject in need thereof, in
particular a subject such as a human having an abnormality in
either ROS1 gene or ALK gene, in particular a ROS1 chromosome
rearrangement or an ALK chromosome rearrangement.
Namely the method of treating a subject suffering from cancer
comprises administering an effective amount of a compound having
the structure of Formula (Ia) or a pharmaceutically acceptable
salt, solvate or anhydrate thereof to the subject:
##STR00001## R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are each
independently selected from hydrogen, halogen, C.sub.1-C.sub.3
haloalkyl, nitro, cyano or C.sub.1-C.sub.3-alkyl. R.sup.5
represents a hydrophobic moiety and is selected from optionally
substituted C.sub.6-C.sub.10-aryl or optionally substituted
C.sub.7-C.sub.10-aralkyl. R.sup.6, R.sup.7, R.sup.8, R.sup.9 and
R.sup.10 are each independently selected from hydrogen,
C.sub.1-C.sub.3-alkoxy or C.sub.1-C.sub.3-alkylamino, with the
provisio that at least one of R.sup.6, R.sup.7, R.sup.8, R.sup.9
and R.sup.10 is selected from C.sub.1-C.sub.3-alkoxy or
C.sub.1-C.sub.3-alkylamino, in particular at least R.sub.8 is
selected from C.sub.1-C.sub.3-alkoxy or C.sub.1-C.sub.3-alkylamino.
n and m are each an integer, wherein n is selected from 0, 1, 2 or
3 and m is selected from 0, 1, 2, or 3, wherein the sum of n and m
is at least 1 and at most 4.
Hence, the compound of the present invention comprises certain
structural components, namely a quinoline moiety in the backbone,
i.e. the core part of the compound, at least one phenyl moiety with
at least one hydrophobic substituent, which phenyl moiety is
attached to the backbone via an at most 6-membered linking group
and a further hydrophobic moiety attached to the backbone, namely
R.sub.5. The inventors found that such compound of Formula (Ia) is
especially suitable for inhibiting ROS1 kinase activity, in
particular ROS1 fusion kinase activity. Moreover, the inventors
found that the compound of Formula (Ia) having the above mentioned
structural components is also exceptionally effective in inhibiting
ALK kinase activity, in particular ALK fusion kinase activity.
In particular, the compound has the structure of Formula (Ic):
##STR00002##
In still another aspect, the present invention refers to a method
of inhibiting ROS1 kinase activity, in particular ROS1 fusion
kinase activity, or ALK kinase activity, in particular ALK fusion
kinase activity, in cancer cells by a compound of Formula (Ia) in a
subject in need thereof, i.e. comprising administering an effective
amount of the compound of Formula (Ia), in particular of Formula
(Ic), to a subject suffering from cancer, in particular lung cancer
like NSCLC. In one embodiment the disease is ROS1-dependent NSCLC.
In another embodiment, the disease is ALK-dependent NSCLC.
According to the invention is also the compound of Formula (Ia)
such as Formula (Ic) for use as a medicament, preferably for use in
the treatment of cancer such as NSCLC like ROS1-dependent or
ALK-dependent NSCLC. Furthermore, the invention refers to the use
of the compound of Formula (Ia) such as Formula (Ic) for preparing
a medicament for treatment of a disease, in particular cancer such
as NSCLC like ROS1-dependent NSCLC or ALK-dependent NSCLC.
Another aspect of the present invention relates to a composition
comprising the compound of Formula (Ia) or a salt, solvate or
anhydrate thereof:
##STR00003##
Wherein R.sup.1 to R.sup.10 and n and m are as defined above. In
particular the composition is a pharmaceutical composition
comprising the compound of Formula (Ia) or a pharmaceutically
acceptable salt, solvate or anhydrate thereof. Said pharmaceutical
composition further comprises physiologically tolerable excipients
and may additionally contain further active ingredients, in
particular therapeutic compounds for treating cancer such as NSCLC.
The present invention also refers to the use of the composition for
inhibiting ROS1 kinase activity or ALK kinase activity, in
particular ROS1 fusion kinase activity or ALK fusion kinase
activity, such as for suppressing phosphorylation of ROS1 kinase or
ALK kinase, in particular ROS1 fusion kinase or ALK fusion kinase,
and/or inhibiting the anti-apoptotic and growth signaling
downstream to ROS1 kinase or ALK kinase, in particular ROS1 fusion
kinase or ALK fusion kinase.
The present invention, in another aspect, refers to a method for
targeting cancer cells harboring an abnormality in ROS1 gene or an
abnormality in ALK gene, in particular an abnormality in ROS1 gene
resulting from a ROS1 chromosome rearrangement such as those
associated with the expression of at least one ROS1 fusion kinase
including SLC34A2-ROS1 or CD74-ROS1 or an abnormality in ALK gene
resulting from an ALK chromosome rearrangement such as those
associated with the expression of at least one ALK fusion kinase
including EML4-ALK fusion kinases. Said method of the present
invention comprises the step of contacting said cells with a
compound of Formula (Ia) or a salt, solvate or anhydrate
thereof:
##STR00004##
R.sup.1 to R.sup.10 and n and m are as defined above.
Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described. The invention includes all
such variations and modifications. The invention also includes all
steps and features referred to or indicated in the specification,
individually or collectively, and any and all combinations of the
steps or features.
Other features and aspects of the invention will become apparent by
consideration of the following detailed description and
accompanying drawings.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1A shows the cell viability of HCC78 cells after 72 hours
treatment with the compound of Formula (Ic).
FIG. 1B shows the cell viability of BEAS-2B cells after 72 hours
treatment with the compound of Formula (Ic).
FIG. 2A to 2E show fluorescence images of HCC78 cells having been
treated with different concentrations of the compound of Formula
(Ic), with crizotinib or the control group. FIG. 2A shows a
fluorescence image of HCC78 cells having been treated with 2.5
.mu.M crizotinib. FIG. 2B shows a fluorescence image of the control
group of HCC78 cells. FIG. 2C shows a fluorescence image of HCC78
cells having been treated with 1.25 .mu.M of the compound of
Formula (Ic). FIG. 2D shows a fluorescence image of HCC78 cells
having been treated with 2.5 .mu.M of the compound of Formula (Ic).
FIG. 2E shows a fluorescence image of HCC78 cells having been
treated with 5 .mu.M of the compound of Formula (Ic).
FIG. 3A to 3E show a Flow Cytometry pattern of HCC78 cells having
been treated with different concentrations of the compound of
Formula (Ic), with crizotinib or of the control group. FIG. 3A
shows a Flow Cytometry pattern of HCC78 cells having been treated
with 2.5 .mu.M crizotinib. FIG. 3B shows a Flow Cytometry pattern
of the control group of HCC78 cells. FIG. 3C shows a Flow Cytometry
pattern of HCC78 cells having been treated with 1.25 .mu.M of the
compound of Formula (Ic). FIG. 3D shows a Flow Cytometry pattern of
HCC78 cells having been treated with 2.5 .mu.M of the compound of
Formula (Ic). FIG. 3E shows a Flow Cytometry pattern of HCC78 cells
having been treated with 5 .mu.M of the compound of Formula
(Ic).
FIG. 3F shows the rate of apoptosis of HCC78 cells having been
treated with the compound of Formula (Ic) of the present invention
(referenced as "G341-0312") with 1.25 .mu.M, 2.5 .mu.M or 5 .mu.M
or 2.5 .mu.M crizotinib compared to the control group.
FIG. 4 refers to a western blot and shows the expression of
phosphorylated ROS1, ROS1, phosphorylated AKT, AKT, phosphorylated
STAT3, STAT3, phosphorylated ERK, ERK and GAPDH of a control group
and HCC78 cells treated with 2.5 .mu.M crizotinib, 1.25 .mu.M, 2.5
.mu.M or 5 .mu.M of the compound of Formula (Ic) (referenced as
"G341-0312").
FIG. 5A shows a 3D schematic representation of the compound of
Formula (Ic), crizotinib and the binding pocket of the ROS1 kinase
domain.
FIG. 5B shows a 3D schematic representation of the binding mode
between the compound of Formula (Ic) and the binding pocket of the
ROS1 kinase domain.
FIG. 6A shows the cell viability of H2228 cells after 72 hours
treatment with the compound of Formula (Ic) of the present
invention.
FIG. 6B shows the cell viability of BEAS-2B cells after 72 hours
treatment with the compound of Formula (Ic) of the present
invention.
FIG. 7A to 7E show a Flow Cytometry pattern of H2228 cells having
been treated with different concentrations of the compound of
Formula (Ic) of the present invention, with crizotinib or of the
control group. FIG. 7A shows a Flow Cytometry pattern of H2228
cells having been treated with 5 .mu.M crizotinib. FIG. 7B shows a
Flow Cytometry pattern of the control group of H2228 cells. FIG. 7C
shows a Flow Cytometry pattern of H2228 cells having been treated
with 1.25 .mu.M of the compound of Formula (Ic). FIG. 7D shows a
Flow Cytometry pattern of H2228 cells having been treated with 2.5
.mu.M of the compound of Formula (Ic). FIG. 7E shows a Flow
Cytometry pattern of H2228 cells having been treated with 5 .mu.M
of the compound of Formula (Ic).
FIG. 7F shows the rate of apoptosis of H2228 cells having treated
with the compound of Formula (Ic) of the present invention
(referenced as "G341-0312") with 1.25 .mu.M, 2.5 .mu.M or 5 .mu.M
or with 5 .mu.M crizotinib compared to the control group.
FIG. 8A to 8E show the formation of H2228 cell colonies after
treatment with different concentrations of the compound of Formula
(Ic), crizotinib or of the control group. FIG. 8A refers to the
formation of H2228 cell colonies after treatment with 5 .mu.M
crizotinib. FIG. 8B refers to the formation of H2228 cell colonies
in the control group. FIG. 8C refers to the formation of H2228 cell
colonies after treatment with 1.25 .mu.M of the compound of Formula
(Ic). FIG. 8D refers to the formation of H2228 cell colonies after
treatment with 2.5 .mu.M of the compound of Formula (Ic). FIG. 8E
refers to the formation of H2228 cell colonies after treatment with
5 .mu.M of the compound of Formula (Ic).
FIG. 8F illustrates the average number of colonies formed in the
colony formation assay as shown in FIG. 8A to 8E, i.e. with 1.25
.mu.M, 2.5 .mu.M and 5 .mu.M of the compound of Formula (Ic)
(referenced as "G341-0312") compared with 5 .mu.M crizotinib and
control group.
FIG. 9 refers to a western blot and shows the expression of
phosphorylated ALK, ALK, phosphorylated AKT, AKT, phosphorylated
STAT3, STAT3, phosphorylated ERK, ERK and GAPDH of a control group
and H2228 cells treated with 5 .mu.M crizotinib, with 1.25 .mu.M,
2.5 .mu.M or 5 .mu.M of compound of Formula (Ic) (referenced as
"G341-0312").
FIG. 10A shows a 3D schematic representation of the compound of
Formula (Ic), crizotinib and the binding pocket of the ALK kinase
domain.
FIG. 10B shows a 3D schematic representation of the binding mode
between the compound of Formula (Ic) and the binding pocket of the
ALK kinase domain.
DETAILED DESCRIPTION OF INVENTION
Unless otherwise defined, all technical terms used herein have the
same meaning as commonly understood by one skilled in the art to
which the invention belongs unless indicated otherwise.
The present invention provides a compound for use in a method for
treating cancer in a subject in need thereof. More specifically,
the present invention, in a first aspect, refers to a method of
treating cancer by a compound in a subject in need thereof, namely
a method of treating a subject suffering from cancer comprising
administering an effective amount of a compound to the subject. The
cancer is, in particular, a NSCLC such as a NSCLC adenocarcinoma,
in particular ROS1-dependent NSCLC or ALK-dependent NSCLC.
The term "ROS1-dependent" (or ROS1-positive) as used within this
patent application refers to a cancer with cancer cells harboring
an abnormality in ROS1 gene. An abnormality in ROS1 gene preferably
results from a ROS1 chromosome rearrangement, also referenced as
ROS1 gene fusion. "ROS1 chromosome rearrangement" used herein
refers to a type of chromosome abnormality such as due to
interchromosomal translocation or intrachromosomal deletion,
inversion or duplication involving the ROS1 gene, which results in
the creation of fusion genes of the rearrangement partner and the
ROS1 gene or parts thereof usually associated with the expression
of ROS1 fusion kinases containing the whole kinase domain of ROS1
wild-type kinase.
The abnormality in ROS1 gene preferably results from a ROS1
chromosome rearrangement selected from at least one of SLC34A2 (or
SCL34A2)-ROS1, CD74-ROS1, CLTC-ROS1, EZR-ROS1, TPM3-ROS1,
SDC4-ROS1, LRIG3-ROS1, KDELR2-ROS1, CCDC6-ROS1, LIMA1-ROS1,
FIG-ROS1 or MSN-ROS1, more preferably SLC34A2-ROS1 or CD74-ROS1.
This also includes respective variants of the aforementioned
chromosome rearrangements. Preferably, said abnormality in ROS1
gene is associated with a detectable expression of a ROS1 kinase,
if the ROS1 kinase is not expressed in noncancerous cells without
abnormality of ROS1 gene of the same cell type, otherwise an
increase in the expression of a ROS1 kinase compared to
non-cancerous cells of the same cell type. The abnormality in ROS1
gene preferably results from a ROS1 chromosome rearrangement
associated with a detectable expression of at least one ROS1 fusion
kinase selected from the group consisting of SLC34A2 (or
SCL34A2)-ROS1 (including SLC34A2-ROS1(S), SLC34A2-ROS1(L) and
SLC34A2-ROS1(VS)), CD74-ROS1, CLTC-ROS1, EZR-ROS1, TPM3-ROS1,
SDC4-ROS1, LRIG3-ROS1, KDELR2-ROS1, CCDC6-ROS1, LIMA1-ROS1,
FIG-ROS1 (including FIG-ROS1(L), FIG-ROS1(S) and FIG-ROS1(VL)) and
MSN-ROS1, more preferably selected from the group consisting of
SLC34A2-ROS1 and CD74-ROS1 fusion kinases. In all these fusion
kinases, the ROS1 kinase domain of ROS1 wild-type kinase is fully
retained. I.e. ROS1-dependent cancer or subjects preferably have a
detectable expression of at least one ROS1 fusion kinase,
respectively, as a result of the fusion between ROS1 gene and
another rearrangement gene.
Accordingly, the term "ALK-dependent" (or ALK-positive) as used
herein refers to a cancer with cancer cells harboring an
abnormality in ALK gene. The abnormality in ALK gene preferably
results from one or more of: an ALK chromosome rearrangement,
additional gene copies of the ALK gene or point mutations in the
ALK gene itself in particular point mutations in the tyrosine
kinase domain, i.e. mutations affecting only one or very few
nucleotides in the ALK gene sequence. "ALK chromosome
rearrangement" used herein refers to a type of chromosome
abnormality such as due to interchromosomal translocation or
intrachromosomal deletion, inversion or duplication involving the
ALK gene, which results in the creation of fusion genes of the
rearrangement partner and the ALK gene usually associated with the
expression of ALK fusion kinase containing the whole kinase domain
of ALK wild-type kinase.
Most preferably, said abnormality in ALK gene is an ALK chromosome
rearrangement, also referenced as ALK gene fusion. Hence, most
preferably ALK-dependent means cancer with cells harboring an
abnormality in ALK gene, which abnormality in ALK gene results from
an ALK chromosome rearrangement. The chromosome rearrangement is,
preferably, selected from one or more of EML4-ALK, KIF5B-ALK,
KLC1-ALK, PTPN3-ALK, STRN-ALK and TFG-ALK, most preferably
EML4-ALK. This also includes respective variants of the
aforementioned chromosome rearrangements in particular variants of
EML4-ALK chromosome rearrangements which include, for example,
EML4-ALK, E13;A20 (variant 1), EML4-ALK, E20;A20 (variant 2),
EML4-ALK, E6a/b;A20 (variant 3a/b), EML4-ALK, E14;A20 (variant 4),
EML4-ALK, E2a/b;A20 (variant 5a/b), EML4-ALK, E13b;A20 (variant 6),
EML4-ALK, E14;A20 (variant 7), EML4-ALK, E15;A20 (variant "V4"),
EML4-ALK, E17;A20 and EML4-ALK, E18;A20 (variant "V5"). Variants of
KIF5B-ALK include, for example, KIF5B-ALK, K17;A20 or KIF5B-ALK,
K24;A20.
Preferably, the abnormality in ALK gene is associated with a
detectable expression of an ALK kinase, if the ALK kinase is not
expressed in noncancerous cells without abnormality of ALK gene of
the same cell type, otherwise an increase in the expression of an
ALK kinase compared to non-cancerous cells of the same cell type.
Especially preferably, said abnormality in ALK gene is an ALK
chromosome rearrangement associated with a detectable expression of
at least one ALK fusion kinase in particular selected from the
group consisting of EML4-ALK, KIF5B-ALK, KLC1-ALK, PTPN3-ALK,
STRN-ALK and TFG-ALK. Most preferably selected from of at least one
EML4-ALK fusion kinase in particular at least one EML4-ALK fusion
kinase resulting from a variant of EML4-ALK chromosome
rearrangement including EML4-ALK, E13;A20 (variant 1), EML4-ALK,
E20;A20 (variant 2), EML4-ALK, E6a/b;A20 (variant 3a/b), EML4-ALK,
E14;A20 (variant 4), EML4-ALK, E2a/b;A20 (variant 5a/b), EML4-ALK,
E13b;A20 (variant 6), EML4-ALK, E14;A20 (variant 7), EML4-ALK,
E15;A20 (variant "V4"), EML4-ALK, E17;A20 and EML4-ALK, E18;A20
(variant "V5"), in particular from EML4-ALK, E13;A20 (variant 1),
EML4-ALK, E20;A20 (variant 2) or EML4-ALK, E6a/b;A20 (variant
3a/b).
In all these fusion kinases, the ALK kinase domain of ALK wild-type
kinase is fully retained. I.e. ALK-dependent cancer or subjects
preferably have a detectable expression of at least one ALK fusion
kinase, respectively, as a result of the fusion between the ALK
gene and another gene.
An "increased expression" of ROS1 kinase or ALK kinase means an
expression at least 5% and preferably at least 10% higher than in
the control group, i.e. non-cancerous cells without abnormality of
ROS1 or ALK gene. The skilled person is aware of suitable methods
for determining ROS1 kinase or ALK kinase expression.
ROS1 wild-type kinase or ALK wild-type kinase, its structure as
well as ROS1 chromosome rearrangements or ALK chromosome
rearrangements and gene fusions, respectively, as well as resulting
ROS1 fusion kinases and ALK fusion kinases are known to the skilled
person. "ROS1 wild-type kinase" (or -protein) and "ALK wild-type
kinase" (or -protein) generally refer to the respective full length
protein with the sequence as encoded in normal (healthy) cells or
tissue, namely non-cancerous cells or tissue, i.e. without ROS1 or
ALK involving chromosome rearrangements. In contrast, "ROS1 fusion
kinase" and "ALK fusion kinase" refer to the fusion protein
expressed after ROS1 involving chromosome rearrangement or ALK
involving chromosome rearrangement, in which at least the kinase
domain of the ROS1 wild-type protein or ALK wild-type protein fused
to all or a portion of another protein and polypeptide,
respectively. For example, SLC34A2-ROS1 is a fusion of a portion of
the SLC34A2 polypeptide with a portion of the ROS1 polypeptide
based on a gene fusion of respective encoding polynucleotides. The
CD74 gene encodes a type 2 transmembrane protein that fuses with
ROS1 to generate a CD74-ROS1 transcript found to be the most common
form of all ROS1 fusion genes in NSCLC, accounting for about 40% of
all ROS1 fusions genes in NSCLC. The terms "ROS1 kinase" and "ALK
kinase" generally cover wild-type kinases as well as fusion
kinases.
Whether a cancer or a subject is ROS1-dependent or ALK-dependent
can be confirmed by respective molecular biological methods,
wherein several methods are known to the skilled person. Commonly
used and suitable methods especially include fluorescence in situ
hybridization (FISH) (i.e. Shaw, A. T. et al., Nature 2013,
13:772-787), immunohistochemistry (IHC) (i.e. Shaw, A. T. et al.,
Nature 2013, 13:772-787) and quantitative real-time reverse
transcription-PCR (qRT-PCR) assays or chromogenic in situ
hybridization (CISH) (Gandhi, L. and Jaenne, P. A., Clinical cancer
research, 2012, 18:3737-3742). I.e. "ROS1-dependent cancer" or
"abnormality in ROS1 gene" is in particular considered for being
present when at least one of the methods selected from FISH, IHC,
CISH or qRT-PCR assay reveals a ROS1 chromosome rearrangement.
Accordingly, "ALK-dependent cancer" or "abnormality in ALK gene" is
in particular considered for being present when at least one of the
methods selected from FISH, IHC, CISH or qRT-PCR assay reveals an
ALK chromosome rearrangement. The same is true with regard to the
specific type of ROS1 or ALK chromosome rearrangement, for which
methods, in particular fusion partner specific assays, are known to
the skilled person, as well.
The cancer is preferably a lung cancer, in particular a
ROS1-dependent lung cancer or an ALK-dependent lung cancer.
Preferably, the lung cancer is NSCLC. Hence, in especially
preferred embodiments of the present invention, the disease is
NSCLC, in particular a ROS1-dependent NSCLC or an ALK-dependent
NSCLC. The disease is, in particular, NSCLC adenocarcinoma.
The terms "cancer" and "cancerous" refer to or describe a
physiological condition in subjects in which a population of cells
are characterized by unregulated cell growth. The term "tumor"
simply refers to a mass being of benign (generally harmless) or
malignant (cancerous) growth.
The method of the present invention comprises administering an
effective amount of a compound or a pharmaceutically acceptable
salt, solvate or anhydrate thereof to a subject. The subject can be
a human or animal, in particular the subject is a human. In
preferred embodiments of the present invention, the subject is a
mammal having an abnormality in ROS1 gene resulting from ROS1
chromosome rearrangement, which preferably includes ROS1 chromosome
rearrangement with the generation of at least one of SLC34A2-ROS1
or CD74-ROS1 fusion kinase. The inventors found that the compound
is sufficiently effective in treating subjects with abnormality in
ALK gene, as well. Hence, in another embodiment of the present
invention, the subject is a mammal having an abnormality in ALK
gene resulting from an ALK chromosome rearrangement, which
preferably includes ALK chromosome rearrangement with the
generation of at least one EML4-ALK fusion kinase, i.e. at least
one fusion kinase resulting from a EML4-ALK gene fusion including
respective variants, in particular at least one fusion kinase
selected from EML4-ALK, E13;A20 (variant 1), EML4-ALK, E20;A20
(variant 2) or EML4-ALK, E6a/b;A20 (variant 3a/b).
The compound of the present invention has a structure of Formula
(Ia):
##STR00005##
R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are each independently
selected from hydrogen, halogen, C.sub.1-C.sub.3 haloalkyl, nitro,
cyano or C.sub.1-C.sub.3-alkyl. In preferred embodiments, R.sup.1,
R.sup.2, R.sup.3 and R.sup.4 are each independently selected from
hydrogen, halogen or C.sub.1-C.sub.2 haloalkyl. In further
preferred embodiments, R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are
each independently selected from hydrogen, Cl, Br or F, in
particular from hydrogen or Cl. In especially preferred embodiments
of the present invention, R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are
all hydrogen.
R.sup.5 represents a hydrophobic moiety. R.sub.5 is selected from
optionally substituted C.sub.6-C.sub.10-aryl or optionally
substituted C.sub.7-C.sub.10-aralkyl. Preferably, R.sup.5 is
selected from C.sub.6-C.sub.10-aryl or C.sub.7-C.sub.10-aralkyl,
still more preferably from a C.sub.6-C.sub.10-aryl. In particular
embodiments of the present invention, R.sup.5 is optionally
substituted C.sub.6-C.sub.10-aryl, i.e. C.sub.6-C.sub.10-aryl which
may contain further substituents, namely at least one hydrogen
atom, in particular one hydrogen atom, is replaced with a
substituent, for example, a C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3
alkoxy, C.sub.1-C.sub.3 alkylamino, preferably a C.sub.1-C.sub.2
alkyl, C.sub.1-C.sub.2 alkoxy or a C.sub.1-C.sub.2 alkylamino.
Still more preferably, R.sup.5 is optionally substituted phenyl,
wherein at least one hydrogen atom, in particular one hydrogen
atom, is optionally replaced with a substituent, preferably a
C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3 alkoxy or a C.sub.1-C.sub.3
alkylamino, more preferably a C.sub.1-C.sub.2 alkyl,
C.sub.1-C.sub.2 alkoxy or a C.sub.1-C.sub.2 alkylamino, still more
preferably a C.sub.1-C.sub.2 alkoxy. Still more preferably, R.sup.5
is a moiety having the structure:
##STR00006##
In said embodiments, R is hydrogen, a C.sub.1-C.sub.2 alkyl,
C.sub.1-C.sub.2 alkoxy or a C.sub.1-C.sub.2 alkylamino. Most
preferably, R is hydrogen, i.e. the phenyl is unsubstituted.
Accordingly, in especially preferred embodiments of the present
invention, R.sup.5 is C.sub.6-C.sub.10-aryl, i.e. unsubstituted
C.sub.6-C.sub.10-aryl, and in particular phenyl, i.e. unsubstituted
C.sub.6 aryl.
R.sup.6, R.sup.7, R.sup.8, R.sup.9 and R.sup.10 are each
independently selected from hydrogen, C.sub.1-C.sub.3-alkoxy or
C.sub.1-C.sub.3-alkylamino, with the provisio that at least one of
R.sup.6, R.sup.7, R.sup.8, R.sup.9 and R.sup.10 is selected from
C.sub.1-C.sub.3-alkoxy or C.sub.1-C.sub.3-alkylamino, preferably at
least R.sub.8 is selected from C.sub.1-C.sub.3-alkoxy or
C.sub.1-C.sub.3-alkylamino. Still more preferably, R.sup.6,
R.sup.7, R.sup.8, R.sup.9 and R.sup.10 are each independently
selected from hydrogen or C.sub.1-C.sub.3-alkoxy, with the provisio
that at least one of R.sup.6, R.sup.7, R.sup.8, R.sup.9 and
R.sup.10 is selected from C.sub.1-C.sub.3-alkoxy. In particular,
R.sup.6, R.sup.7, R.sup.8, R.sup.9 and R.sup.10 are each
independently selected from hydrogen or C.sub.1-C.sub.2-alkoxy,
with the provisio that at least one of R.sup.6, R.sup.7, R.sup.8,
R.sup.9 and R.sup.10 is C.sub.1-C.sub.2-alkoxy. More preferably,
R.sup.6, R.sup.7, R.sup.8, R.sup.9 and R.sup.10 are each
independently selected from hydrogen or methoxy, with the provisio
that at least one of R.sup.6, R.sup.7, R.sup.8, R.sup.9 and
R.sup.10 is methoxy. In an especially preferred embodiment of the
present invention, R.sup.6, R.sup.7, R.sup.9 and R.sup.10 are
hydrogen and R.sup.8 is C.sub.1-C.sub.2-alkoxy, in particular
methoxy.
n and m are an integer and each indicate the number of methylene
groups. For example, if n is 0, there is no methylene group present
at the respective position, i.e. the carbonyl-group is directly
bonded to the nitrogen atom in the pyrazole ring structure. In case
of n=1, there is one methylene group connecting the carbonyl group
with the nitrogen atom in the pyrazole ring structure. n is
selected from 0, 1, 2 or 3 and m is selected from 0, 1, 2 or 3,
wherein the sum of n and m is at least 1 and at most 4. More
preferably, the sum of n and m is at most 3 and in particular at
most 2, most preferably the sum of n and m is 2. n is preferably
selected from 0, 1 or 2 and m is preferably selected from 1, 2 or
3. In especially preferred embodiments, n and m are both 1.
The term "optionally substituted" as used herein means that said
radical or group is either unsubstituted or substituted.
"Substituted" means that one or more hydrogen atoms of that radical
or group, preferably one to two hydrogen atoms, in particular one
hydrogen atom, are replaced with certain substituents provided that
the normal valency is not exceeded and that the substitution
results in a chemically stable compound. For example, optionally
substituted C.sub.7-C.sub.10-aralkyl means that the radial may be
substituted at the aryl ring or not. Use of the term
C.sub.7-C.sub.10-aralkyl without the expression "optionally
substituted" is to be understood to refer to unsubstituted
C.sub.7-C.sub.10-aralkyl, only. This also applies with regard to
the term "optionally substituted C.sub.6-C.sub.10-aryl" versus
"C.sub.6-C.sub.10-aryl".
The term "C.sub.1-C.sub.3 alkyl" as group used in the present
invention refers to a hydrocarbyl radical having from 1 to 3 carbon
atoms which includes a straight chain or branched alkyl group.
Namely, it comprises methyl, ethyl, propyl and isopropyl. Likewise,
"C.sub.1-C.sub.2 alkyl" refers to a hydrocarbyl radical having 1 to
2 carbon atoms.
"C.sub.1-C.sub.3 alkoxy" refers to a radical having a formula -AB
wherein A is an oxygen atom and B is C.sub.1-C.sub.3 alkyl, i.e.
including methoxy, ethoxy, propoxy and isopropyloxy.
"C.sub.1-C.sub.2 alkoxy" refers to a radical having a formula -AB
wherein A is an oxygen atom and B is C.sub.1-C.sub.2 alkyl, i.e.
including methoxy and ethoxy.
The term "C.sub.1-C.sub.3 alkylamino" refers to a radical having a
formula --NB.sub.xH.sub.y, wherein x and y are selected from among
x=1, y=1 and x=2, y=0. B is a C.sub.1-C.sub.3 alkyl, i.e. the
number of carbon atoms in B is 1 to 3. When x=2, the total number
of carbon atoms of both B groups is from 1 to 3. "C.sub.1-C.sub.3
alkylamino" includes N-methylamino-, N,N-dimethylamino- and
N-ethylamino- or N-ethyl-N-methylamino-. "C.sub.1-C.sub.2
alkylamino" likewise refers to a radical having a formula
--NB.sub.xH.sub.y, wherein x and y are as defined above and B is a
C.sub.1-C.sub.2 alkyl, i.e. the number of carbon atoms in B is 1 to
2.
The term "C.sub.7-C.sub.10 aralkyl" refers to an alkyl radical with
an aryl ring, i.e. a radical having a formula -AB, wherein A is a
branched or straight chain hydrocarbyl radical and B is an aryl
ring, usually a phenyl ring, attached to the hydrocarbyl radical,
both in total comprise from 7 to 10 carbon atoms. Examples of such
groups include benzyl (i.e. phenylmethyl), phenethyl, phenylpropyl,
phenylbutyl. The "C.sub.7-C.sub.10 aralkyl" is optionally
substituted, i.e. at least one hydrogen atom, in particular one
hydrogen atom, may be replaced with a substituent, for example,
selected from a C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3 alkoxy or
C.sub.1-C.sub.3 alkylamino.
"C.sub.6-C.sub.10 aryl" according to the invention means an
hydrocarbon residue with 6 to 10 carbon atoms having a ring with a
maximum number of double bonds, i.e. the maximum number of u
electrons, in particular an aromatic ring, and includes monocyclic
and polycyclic hydrocarbons wherein the additional ring(s) of the
polycyclic hydrocarbon may be saturated, i.e. without double or
triple bonds, unsaturated or aromatic. "Unsaturated" means the
presence of at least one double or triple bond. "Aromatic" means
the presence of a delocalized, conjugated .pi.-electron system,
namely the term "aromatic" generally means a ring having a
delocalized .pi.-electron system containing 4n+2.pi. electrons,
where n is an integer and at least 0. Most preferably,
C.sub.6-C.sub.10 aryl refers to an aromatic hydrocarbon with 6 to
10 carbon atoms, more preferably with 6 carbon atoms. The
C.sub.6-C.sub.10 aryl is optionally substituted; i.e. it may
contain further substituents, namely at least one hydrogen atom, in
particular one hydrogen atom, is replaced with a substituent, for
example, a C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3 alkoxy or
C.sub.1-C.sub.3 alkylamino, preferably a C.sub.1-C.sub.2 alkyl,
C.sub.1-C.sub.2 alkoxy or a C.sub.1-C.sub.2 alkylamino. Examples of
"C.sub.6-C.sub.10 aryl" include phenyl, indenyl and naphthyl.
"Nitro" refers to a --NO.sub.2 group, wherein "cyano" refers to a
--CN group. The term "halogen" as a group or part of a group refers
to fluoro (--F), chloro (--Cl), bromo (--Br), iodo (--I) unless
otherwise indicated.
The term "C.sub.1-C.sub.3 haloalkyl" as used herein as a group
refers to a straight chain or branched alkyl group with 1 to 3
carbon atoms, wherein one or more hydrogen atoms are replaced with
a halogen, in particular one hydrogen atom is substituted with a
halogen. Examples of such groups include fluoroethyl, fluoromethyl,
trifluoromethyl or trifluoroethyl and the like.
Also contemplated by the present invention are any pharmaceutically
acceptable salts, anhydrates, solvates, anhydrates as well as
enantiomers and their mixtures, stereoisomeric forms, racemates,
diastereomers and their mixtures of the compound of Formula
(Ia).
As used herein, the term "solvate" refers to a complex of variable
stoichiometry formed by a solute, i.e. compound of Formula (Ia),
and a solvent. If the solvent is water, the solvate formed is a
hydrate. As used herein, the term "anhydrate" means any compound
free of the water of hydration, as would be understood in the art.
Suitable pharmaceutically acceptable salts are those which are
suitable to be administered to subjects, in particular mammals such
as humans and can be prepared with sufficient purity and used to
prepare a pharmaceutical composition. The terms enantiomers,
stereoisomeric forms, racemates, diastereomers are known to the
skilled person.
Said compound of Formula (Ia) is, amongst others, characterized by
certain structural components, which were found to unexpectedly
contribute to an advantageous inhibition of the activity of ROS1
receptor tyrosine kinase by interacting with amino acids within the
ROS1 kinase domain and ROS1 binding pocket, respectively. Namely
the compound of the present invention comprises a quinoline moiety
in the backbone, at least one phenyl containing moiety in a side
chain attached to said backbone via an at most 6-membered linking
group having at least one hydrophobic substituent as well as a
hydrophobic moiety attached to the backbone referenced as R.sup.5.
Further moieties which may be attached to the backbone or side
chain according to Formula (Ia) do not impede the interaction of
compound of Formula (Ia) with the ROS1 kinase domain and preferably
allow for additional interactions including van der Waals forces
and hydrogen bonds or hydrophobic interactions with the ROS1 kinase
domain and, thus, further contribute to the exceptional interaction
with ROS1 kinase.
The inventors unexpectedly found that the presence of these
structural components allows for advantageous multiple interactions
with the ROS1 tyrosine kinase domain, in particular the substituted
phenyl moiety in the side chain and R.sup.5 contribute to
advantageous and close hydrophobic interactions with the hinge
region and the G-loop in addition to hydrogen bonds in particular
with e.g. Met2029 formed by the quinoline moiety. These
interactions are considered for being important ones allowing for
the advantageous interaction with the ROS1 kinase domain and a
potent inhibition of the activity of ROS1 kinase and, hence, ROS1
fusion kinases as said ROS1 kinase domain is fully retained in
known fusion partners, i.e. in known ROS1 fusion kinases.
The inventors further found that the compound of Formula (Ia) with
those structural components, such as the compound of Formula (Ic),
is also exceptionally suitable to inhibit ALK kinase activity such
as ALK fusion kinase activity. They found that said compound can
advantageously interact with and, thus, bind to the ALK kinase
domain based on multiple interactions, too. Such interactions
proved to allow for sufficiently and exceptionally inhibiting the
ALK phosphorylation and anti-apoptotic and growth signaling
downstream to ALK, too. In this context, the structural components
of the compound of Formula (Ia) proved to allow for forming
hydrogen bonds, e.g. with Met1199 as backbone residue in the hinge
region, and for further advantageous interactions with the ALK
kinase domain, in particular it fills lipophilic pockets formed by
residues of the hinge region and the catalytic spine.
In especially preferred embodiments, the compound is a compound of
Formula (Ib):
##STR00007##
R is hydrogen, a C.sub.1-C.sub.2 alkyl, a C.sub.1-C.sub.2 alkoxy or
a C.sub.1-C.sub.2 alkylamino. Most preferably, R is hydrogen, i.e.
the phenyl is unsubstituted, i.e. has no further substituent.
R.sup.6, R.sup.7, R.sup.8, R.sup.9 and R.sup.10 are each
independently selected from hydrogen, C.sub.1-C.sub.2-alkoxy or
C.sub.1-C.sub.2-alkylamino, with the provisio that at least one of
R.sup.6, R.sup.7, R.sup.8, R.sup.9 and R.sup.10 is selected from
C.sub.1-C.sub.2-alkoxy or C.sub.1-C.sub.2-alkylamino, preferably at
least R.sub.8 is selected from C.sub.1-C.sub.2-alkoxy or C.sub.r
C.sub.2-alkylamino. In particular, R.sup.6, R.sup.7, R.sup.8,
R.sup.9 and R.sup.10 are each independently selected from hydrogen
or C.sub.1-C.sub.2-alkoxy, with the provisio that at least one of
R.sup.6, R.sup.7, R.sup.8, R.sup.9 and R.sup.10 is
C.sub.1-C.sub.2-alkoxy. More preferably, R.sup.6, R.sup.7, R.sup.8,
R.sup.9 and R.sup.10 are each independently selected from hydrogen
or methoxy, with the provisio that at least one of R.sup.6,
R.sup.7, R.sup.8, R.sup.9 and R.sup.10 is methoxy. In an especially
preferred embodiment, R.sup.6, R.sup.7, R.sup.9 and R.sup.10 are
hydrogen and R.sup.8 is C.sub.1-C.sub.2-alkoxy, in particular
methoxy.
In particular embodiments of the present invention, the compound is
a compound of Formula (Ic):
##STR00008## which is also referenced as "G341-0312" herein and
includes any pharmaceutically acceptable salt, hydrate, solvate,
anhydrate as well as enantiomer and their mixtures, stereoisomeric
form, racemate, diastereomer and their mixtures of the compound of
Formula (Ic).
As further shown below, respective data with HCC78 cell lines with
ROS1 chromosome rearrangement as well as with H2228 cell lines with
ALK chromosome rearrangement further confirm that compound of
Formula (Ic) is especially effective in inhibiting ROS1 kinase
activity and ALK kinase activity. The compound of Formula (Ic)
proved to be highly cytotoxic and selective to cancer cells. It
proved to advantageously target ROS1 fusion kinase and ALK fusion
kinase, respectively, while showing relatively low toxicity to
normal lung cells. In particular, the compound of Formula (Ic)
proved to exceptionally inhibit growth, induce apoptosis and
suppress the phosphorylation of ROS1 fusion kinase while making use
of the NSCLC cell line HCC78, which is a NSCLC cell line
characterized by ROS1-driven activated signaling due to the
presence of the SLC34A2-ROS1 fusion gene. Moreover, the compound of
Formula (Ic) proved to exceptionally inhibit growth, induce
apoptosis and suppress the phosphorylation of ALK fusion kinase
while making use of the NSCLC cell line H2228, which is a NSCLC
cell line characterized by the ALK rearrangement EML4-ALK (variant
3). The compound of Formula (Ic) further proved to be highly
cytotoxic and selective to cancer cells while having little effect
on healthy normal non-cancerous cells.
The expression "effective amount" generally denotes an amount
sufficient to produce therapeutically desirable results, wherein
the exact nature of the result varies depending on the specific
disorder which is treated. When the disorder is cancer, the result
is usually an inhibition or suppression of the proliferation of the
cancer cells, a reduction of cancerous cells or the amelioration of
symptoms related to the cancer cells, in particular inhibition,
reduction or prevention of the proliferation of the cancer cells or
induction of cell death, i.e. apoptosis of the cancer cells.
The effective amount of the compound of Formula (Ia) may depend on
the species, body weight, age and individual conditions and can be
determined by standard procedures such as with cell cultures or
experimental animals. The concentration of the compound of Formula
(Ia), such as the compound of Formula (Ic), effective for treating
the subject may, for example, be at least 1.25 .mu.M, preferably at
least 2.5 .mu.M, in particular at least 5 .mu.M.
The compound of Formula (Ia) has an IC.sub.50 on cancer cells of at
most 10 .mu.M and an IC.sub.50 on normal non-cancerous cells being
at least 1.5 times higher, more preferably 2 times higher, most
preferably at least 2.5 times higher than the IC.sub.50 on cancer
cells.
In embodiments of the present invention, the disease is
ROS1-dependent NSCLC and the subject suffering from NSCLC has an
abnormality in ROS1 gene, respectively, and the compound has an
IC.sub.50 on said NSCLC cells of at most 10 .mu.M and an IC.sub.50
on normal lung cells being at least 2 times higher, preferably at
least 2.5 times higher than the IC.sub.50 on the NSCLC cells.
In other embodiments of the present invention, the compound is a
compound of Formula (Ic) and the disease is ROS1-dependent
NSCLC.
In another embodiment of the present invention, the disease is
ALK-dependent NSCLC and the subject suffering from NSCLC has an
abnormality in ALK gene, respectively, and the compound has an
IC.sub.50 on said NSCLC cells of at most 10 .mu.M and an IC.sub.50
on normal lung cells being at least 2 times higher than the
IC.sub.50 on the NSCLC cells.
In still further embodiments of the present invention, the compound
is a compound of Formula (Ic) and the disease is ALK-dependent
NSCLC.
The method of the present invention may further include steps
carried out before administering the compound of Formula (Ia), such
as compound of Formula (Ic), to the subject comprising: Obtaining a
sample, in particular cancer cells, from the subject; Testing said
sample for the level of expression of at least one ROS1 fusion
kinase or identifying at least one ROS1 chromosome rearrangement
and/or testing said sample for the level of expression of at least
one ALK fusion kinase or identifying at least one ALK chromosome
rearrangement; Optionally correlating the level of ROS1 fusion
kinase and/or ALK fusion kinase with outcome and if conditions are
met, administrating the compound of Formula (Ia), in particular
compound of Formula (Ic), to said subject.
According to the invention is also the compound of Formula (Ia), in
particular the compound of Formula (Ib) or (Ic), for use as a
medicament, preferably for use in the treatment of cancer such as
lung cancer, especially NSCLC, in particular ROS1-dependent cancer
or ALK-dependent cancer, especially ROS1-dependent NSCLC or
ALK-dependent NSCLC. The compound of Formula (Ia), in particular
the compound of Formula (Ib) or (Ic), can be used in an effective
amount for treating a human. Another aspect of the invention refers
to the use of the compound of Formula (Ia), in particular the
compound of Formula (Ib) or (Ic), for preparing a medicament for
treatment of a disease, in particular of cancer, especially lung
cancer, in particular NSCLC, especially ROS1-dependent NSCLC or
ALK-dependent NSCLC.
The compound of Formula (Ia) may be used in combination with other
therapeutic compounds, preferably therapeutic compounds which are
used for treating cancer such as lung cancer, especially NSCLC.
In still another aspect, the present invention refers to a method
for inhibiting ROS1 kinase activity, in particular ROS1 fusion
kinase activity, or ALK kinase activity, in particular ALK fusion
kinase activity, in cancer cells by a compound of Formula (Ia) in a
subject in need thereof, i.e. comprising administering an effective
amount of a compound of Formula (Ia), in particular of Formula (Ib)
or (Ic), to a subject suffering from a disease such as cancer, in
particular lung cancer like NSCLC. In one embodiment of the present
invention, the disease is ROS1-dependent NSCLC. In another
embodiment of the present invention, the disease is ALK-dependent
NSCLC.
A further aspect of the present invention relates to a composition
comprising the compound of Formula (Ia) or a salt, solvate or
anhydrate thereof:
##STR00009##
Wherein R.sup.1 to R.sup.10 and n and m are as defined above. The
composition further comprises excipients such as pharmaceutically
acceptable excipients, a buffer, salt, water or a combination
thereof. In particular the composition is a pharmaceutical
composition comprising the compound of Formula (Ia) or a
pharmaceutically acceptable salt, solvate or anhydrate thereof.
Said pharmaceutical composition further comprises physiologically
tolerable excipients and may additionally contain further active
ingredients, in particular therapeutic compounds for treating
cancer such as NSCLC.
The skilled person is able to select suitable excipients depending
on the form of the pharmaceutical composition and is aware of
methods for manufacturing pharmaceutical compositions as well as
able to select a suitable method for preparing the pharmaceutical
composition depending on the kind of excipients and the form of the
pharmaceutical composition. The pharmaceutical composition
according to the invention can be present in solid, semisolid or
liquid form to be administered by an oral, rectal, topical,
parenteral or transdermal or inhalative route to a subject,
preferably a human.
More preferably, the compound in the composition is a compound of
Formula (Ib):
##STR00010##
R is hydrogen, a C.sub.1-C.sub.2 alkyl, C.sub.1-C.sub.2 alkoxy or a
C.sub.1-C.sub.2 alkylamino. Most preferably, R is hydrogen, i.e.
the phenyl is unsubstituted, i.e. has no further substituent.
R.sup.6, R.sup.7, R.sup.8, R.sup.9 and R.sup.10 are each
independently selected from hydrogen, C.sub.1-C.sub.2-alkoxy or
C.sub.1-C.sub.2-alkylamino, with the provisio that at least one of
R.sup.6, R.sup.7, R.sup.8, R.sup.9 and R.sup.10 is selected from
C.sub.1-C.sub.2-alkoxy or C.sub.1-C.sub.2-alkylamino, preferably at
least R.sub.8 is selected from C.sub.1-C.sub.2-alkoxy or
C.sub.1-C.sub.2-alkylamino. In particular, R.sup.6, R.sup.7,
R.sup.8, R.sup.9 and R.sup.10 are each independently selected from
hydrogen or C.sub.1-C.sub.2-alkoxy, with the provisio that at least
one of R.sup.6, R.sup.7, R.sup.8, R.sup.9 and R.sup.10 is C.sub.r
C.sub.2-alkoxy. More preferably, R.sup.6, R.sup.7, R.sup.8, R.sup.9
and R.sup.10 are each independently selected from hydrogen or
methoxy, with the provisio that at least one of R.sup.6, R.sup.7,
R.sup.8, R.sup.9 and R.sup.10 is methoxy. In an especially
preferred embodiment, R.sup.6, R.sup.7, R.sup.9 and R.sup.10 are
hydrogen and R.sup.8 is C.sub.1-C.sub.2-alkoxy, in particular
methoxy.
Especially preferably, the compound in the composition is a
compound having Formula (Ic) or a salt, solvate or anhydrate
thereof:
##STR00011##
The present invention also refers to the use of the composition
such as the pharmaceutical composition for inhibiting ROS1 kinase
activity or ALK kinase activity, in particular ROS1 fusion kinase
activity or ALK fusion kinase activity, such as for suppressing
phosphorylation of ROS1 kinase or ALK kinase, in particular ROS1
fusion kinase or ALK fusion kinase, and/or inhibiting the
anti-apoptotic and growth signaling downstream to ROS1 kinase or
ALK kinase, in particular ROS1 fusion kinase or ALK fusion
kinase.
The present invention in another aspect refers to a method for
targeting cancer cells harboring an abnormality in ROS1 gene or
harboring an abnormality in ALK gene. Said abnormality in ROS1 gene
is in particular a ROS1 chromosome rearrangement. More preferably,
the cancer cells express at least one ROS1 fusion kinase selected
from SLC34A2-ROS1 or CD74-ROS1. Said abnormality in ALK gene is
preferably an ALK chromosome rearrangement, preferably the cancer
cells express at least one ALK fusion kinase selected from an
EML4-ALK fusion kinase, in particular selected from EML4-ALK,
E13;A20 (variant 1), EML4-ALK, E20;A20 (variant 2) or EML4-ALK,
E6a/b;A20 (variant 3a/b). The cancer cells are preferably from a
lung tumor, more preferably from a NSCLC in particular from a NSCLC
adenocarcinoma.
Said method of the present invention comprises the step of
contacting said cells with a compound of Formula (Ia) or a salt,
solvate or anhydrate thereof:
##STR00012##
R.sup.1 to R.sup.10 and n and m are as defined above.
Preferably, the proliferation of the cancer cells is inhibited,
reduced or prevented or apoptosis of the cancer cells is
induced.
Preferably, the cancer cells are contacted with the compound of
Formula (Ia) for at least 12 h, more preferably for at least 24 h.
The compound of Formula (Ia) is preferably used in a concentration
of at least 1.25 .mu.M, more preferably of at least 2.5 .mu.M and
especially preferably of at least 5 .mu.M for contacting the cells.
The cancer cells contacted with the compound of Formula (Ia) may
comprise between 1.0.times.10.sup.3 cells and 1.0.times.10.sup.6
cells, in particular about 1.0.times.10.sup.6 cells.
The compound of Formula (Ia) has an IC.sub.50 on cancer cells of at
most 10 .mu.M and an IC.sub.50 on normal non-cancerous cells being
at least 1.5 times higher, preferably at least 2 times higher, more
preferably at least 2.5 times higher than the IC.sub.50 on cancer
cells.
In embodiments of the present invention, the cancer cells are from
NSCLC and harbor an abnormality in ROS1 gene being a ROS1
chromosome rearrangement and the compound has an IC.sub.50 on the
cancer cells of at most 10 .mu.M and an IC.sub.50 on normal
non-cancerous lung cells being at least 2 times higher, preferably
at least 2.5 times higher than the IC.sub.50 on the cancer
cells.
In another embodiment of the present invention, the cancer cells
are from NSCLC and harbor an abnormality in ALK gene being an ALK
chromosome rearrangement and the compound has an IC.sub.50 on the
cancer cells of at most 10 .mu.M and an IC.sub.50 on normal
non-cancerous lung cells being at least 2 times higher than the
IC.sub.50 on the cancer cells.
Still more preferably, the compound used for contacting said cells
is a compound having Formula (Ib):
##STR00013##
R is hydrogen, a C.sub.1-C.sub.2 alkyl, a C.sub.1-C.sub.2 alkoxy or
a C.sub.1-C.sub.2 alkylamino. Most preferably, R is hydrogen, i.e.
the phenyl is unsubstituted, i.e. has no further substituent.
R.sup.6, R.sup.7, R.sup.8, R.sup.9 and R.sup.10 are each
independently selected from hydrogen, C.sub.1-C.sub.2-alkoxy or
C.sub.1-C.sub.2-alkylamino, with the provisio that at least one of
R.sup.6, R.sup.7, R.sup.8, R.sup.9 and R.sup.10 is selected from
C.sub.1-C.sub.2-alkoxy or C.sub.1-C.sub.2-alkylamino, preferably at
least R.sub.8 is selected from C.sub.1-C.sub.2-alkoxy or C.sub.r
C.sub.2-alkylamino. In particular, R.sup.6, R.sup.7, R.sup.8,
R.sup.9 and R.sup.10 are each independently selected from hydrogen
or C.sub.1-C.sub.2-alkoxy, with the provisio that at least one of
R.sup.6, R.sup.7, R.sup.8, R.sup.9 and R.sup.10 is
C.sub.1-C.sub.2-alkoxy. More preferably, R.sup.6, R.sup.7, R.sup.8,
R.sup.9 and R.sup.10 are each independently selected from hydrogen
or methoxy, with the provisio that at least one of R.sup.6,
R.sup.7, R.sup.8, R.sup.9 and R.sup.10 is methoxy. In an especially
preferred embodiment, R.sup.6, R.sup.7, R.sup.9 and R.sup.10 are
hydrogen and R.sup.8 is C.sub.1-C.sub.2-alkoxy, in particular
methoxy.
Especially preferably, the compound used for contacting said cells
is a compound having Formula (Ic) or a salt, solvate or anhydrate
thereof:
##STR00014## wherein the concentration of compound (Ic) for
contacting the cells is at least 2.5 .mu.M, in particular at least
5 .mu.M.
The skilled person is able to prepare the compound of Formula (Ia),
in particular of Formula (Ib) or (Ic), with suitable purity and/or
respective compounds are commercially available with sufficient
purity.
EXAMPLES
Example 1
Inhibition of ROS1 Kinase
The efficiency of the compound of Formula (Ic) as inhibitor of ROS1
has been evaluated. First of all, the cytotoxic properties of the
compound of Formula (Ic) with regard to cells with ROS1 chromosome
rearrangement and its selectivity towards those cancer cells have
been analyzed, its efficacy in inducing cell deaths and inhibition
of colony formation in those cells as well as the effects on the
ROS1 phosphorylation and anti-apoptotic and growth signaling
pathways downstream to ROS1. In the below examples, differences are
analyzed by one-way ANOVA.
All statistical analyses are carried out using Graph Prim5.0.
Values of P<0.05 were considered statistically significant.
Example 1A
Cytotoxic Effects of the Compound of Formula (Ic) Towards Cells
with ROS1 Chromosome Rearrangement
To show the highly cytotoxic and selective properties of the
present compound of Formula (Ic), HCC78 NSCLC cells and normal lung
epithelial cells (BEAS-2B) were treated with the compound of
Formula (Ic) and respective effects were observed. HCC78 NSCLC
cells are non-small cell lung cancer cells with a ROS1 gene fusion.
HCC78 cells were obtained from the American Type Culture Collection
(ATCC) and cultured in environment of 5% CO.sub.2 at 37.degree. C.
in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS),
100 units/mL penicillin, and 100 .mu.g/mL streptomycin. The
compound of Formula (Ic) was dissolved in DMSO. Using a MTT assay,
3000 HCC78 or BEAS-2B cells were seeded on 96-well plates, cultured
overnight for cell adhesion, and treated with DMSO or various
concentrations of the compound of Formula (Ic) for 72 h. Three
independent tests were performed. 10 .mu.L of MTT (5 mg/mL; Sigma)
were added to each well, and incubation continued for another 4 h.
Then the dark blue crystals were dissolved in 100 .mu.L of the
resolved solution (10% SDS and 0.1 mM HCL). The absorbance was
measured at 570 nm by a microplate reader (Tecan, Morrisville,
N.C., USA).
The cell viability was calculated relative to untreated controls,
with results based on at least three independent experiments. The
MTT assay showed that the compound of Formula (Ic) is a potent
inhibitor of HCC78 cells with a IC.sub.50 of 1.62 .mu.M.+-.0.39
.mu.M, while it showed much lower cytotoxicity on normal lung
epithelial cells (BEAS-2B) after 72 h treatment (FIG. 1A and FIG.
1B and table 1). The compound of Formula (Ic) exerts a 3.75-fold
cytotoxicity towards cancer cells than normal healthy cells. The
present compound of Formula (Ic), thus, proved to be highly
selectively towards cancer cells.
TABLE-US-00001 TABLE 1 IC.sub.50 of the compound of Formula (Ic)
Cell lines IC.sub.50 (.mu.M) HCC78 1.62 .mu.M .+-. 0.39 BEAS-2B
6.08 .mu.M .+-. 1.16
Example 1B
Induction of Apoptosis in HCC78 Cells by the Compound of Formula
(Ic)
The compound of Formula (Ic) of the present invention, as a potent
ROS1 inhibitor, proved to induce apoptosis in cancerous cells.
Apoptosis assay was performed on HCC78 cells to demonstrate the
potent ROS1 inhibitory effect of the present invention. HCC78 cells
(1.0.times.10.sup.5 cells/well) were allowed to attach to a 6-well
plate for 24 h, and the cells were treated with the various
concentrations of the compound of Formula (Ic) for additional 72 h.
At the end of incubation, the cells were harvested by
trypsinization and washed twice with ice-cold PBS. After
centrifugation and removal of the supernatants, cell pellets were
resuspended in 100 .mu.L 1.times. Annexin-binding buffer, 2 .mu.L
Annexin-V FITC and 2 .mu.L PI (100 .mu.g/ml) were added and
incubated in the dark at room temperature for 15 min before further
addition of 400 .mu.L of 1.times. Annexin-binding buffer. The
stained cells were analyzed quantitatively using a flow cytometer
(BD Biosciences, San Jose, Calif., USA). FIG. 2A to FIG. 2E show
fluorescence images of HCC78 cells having been treated with the
compound of Formula (Ic) at 1.25 .mu.M, 2.5 .mu.M and 5 .mu.M; 2.5
.mu.M Crizotinib (a known ROS1 inhibitor as positive control) and
DMSO (control, negative control). The results show that HCC78 cells
having been treated with the compound of Formula (Ic) detach from
the surface and are small at 2.5 .mu.M. Such cell morphology
indicates apoptosis.
For a more quantitative view, flow cytometry analysis has been
performed. As evident from FIG. 3F, the present compound of Formula
(Ic) ("G341-0312") exhibits anti-cancer ability through induction
of apoptosis on HCC78 cells in a concentration dependent manner. A
significant apoptosis level is observed in HCC78 cells having been
treated with the compound of Formula (Ic).
Example 1C
Suppression of ROS1 Phosphorylation and Anti-Apoptotic and Growth
Signaling Pathways Downstream to ROS1 by the Compound of Formula
(Ic)
The compound of Formula (Ic) also proved to suppress ROS1
phosphorylation and anti-apoptotic and growth signaling pathways
that are downstream to ROS1. Previous studies demonstrate that ROS1
fusion kinase signal is activated through the tyrosine phosphatase
Src homology-2 domain containing protein tyrosine phosphatase-2
(SHP2) and causes activation of the downstream MEK/ERK,
P13K/AKT/mTOR, and JAK/STAT3 signaling axes. Together, these
downstream signaling pathways promote tumor cell survival and
proliferation. Therefore, inhibition of these downstream signaling
pathways suppresses growth and proliferation of cancer cells and
results in anti-cancer effect.
The cells were planted on 6-well plate, allowed to attach for 24 h,
and treated with the various concentrations of the compound of
Formula (Ic) for 72 h. Cells were washed twice with cold PBS then
lysed in RIPA lysis buffer containing protease and phosphatase
inhibitors. Protein concentration of the cell lysates was measured
using the Bio-Rad protein Assay kit (Bio-Rad, 7 Philadelphia, Pa.,
USA). After equalizing the protein concentrations of the samples,
5.times. laemmli buffer was added and the samples were boiled at
100.degree. C. for 5 min. Equal amounts of protein samples (30
.mu.g) were subjected to SDS-PAGE of a 10% gel. The separated
proteins were transferred to a nitrocellulose (NC) membrane, which
was then exposed to 5% non-fat dried milk in TBS containing 0.1%
Tween (0.1% TBST) for 1 h at room temperature, followed by
overnight incubation at 4.degree. C. with primary antibodies to
GAPDH, phospho-AKT, AKT, phospho-ROS1, ROS1, phospho-ERK, ERK,
phospho-STAT3, STAT3. After washing three times by TBST (5
mins/time), the membranes were incubated for 1 h at room
temperature with the secondary fluorescent antibodies (1:10000
dilutions) to rabbit or mouse. The signal intensity of the
membranes was detected by an LI-COR Odessy scanner (Belfast, Me.,
USA).
Treatment of HCC78 cells with the compound of Formula (Ic)
("G341-0312") led to a dose-dependent decrease of ROS1
phosphorylation as well as of its downstream signaling involving
Erk1/2, STAT3 and AKT, further supporting the anti-cancer effect of
the compound of Formula (Ic) (FIG. 4). Crizotinib has been used as
positive control.
Example 1D
Binding Mode Between the Compound of Formula (Ic) and ROS1
Kinase
The binding mechanism of the compound of Formula (Ic) to ROS1
kinase has been studied. Molecular docking calculation was
performed to study the interaction between compound of Formula (Ic)
and ROS1 kinase by Induced Fit Docking module in Schrodinger
software (Schrodinger, Inc., New York, N.Y., 2009). Compound of
Formula (Ic) was prepared and optimized in the LigPrep module.
During the induced fit docking, centroid of the crizotinib was
defined as the active site and the pose of ligand was valued with
XP docking score. The pose with the highest score was selected for
further analysis. The 3D structure of ROS1 was derived from the PDB
database (PDB ID: 3ZBF) and prepared using the Protein Preparation
Wizard.
The compound of Formula (Ic) proved to have a similar binding
mechanism to ROS1 kinase domain as crizotinib. The docking scores
of the present compound and crizotinib to ROS1 are -11.157 and
-9.674 Kcal/mol, respectively. The present compound is shown to
have a better binding affinity to ROS1 than crizotinib. As seen in
FIG. 5A, the pyridine groups of both compounds form a hydrogen bond
with Met2029 in the hinge region. The phenyl group and the anisole
group of the compound of Formula (Ic) allows for extra hydrophobic
interactions with the hinge and G-loop, respectively. As shown in
FIG. 5B, residues Leu2028, Met2029, Glu2030, Gly2032, Asp2033 in
the hinge region have contact with the compound of Formula (Ic),
while the residues Leu1951, Val1959 in the G-loop have hydrophobic
interactions with the compound of Formula (Ic). Other residues,
such as Lys1980, Asp2102, Leu2086, also highly contribute to the
binding of the compound of Formula (Ic).
Example 2
Inhibition of ALK Kinase
Further, the efficiency of the compound of Formula (Ic) as
inhibitor of ALK kinase has been evaluated including its cytotoxic
properties and selectivity towards cancer cells with ALK chromosome
rearrangement, its efficacy in inducing cell deaths and inhibition
of colony formation in those cells as well as the effects on the
ALK phosphorylation and anti-apoptotic and growth signaling
pathways downstream to ALK.
Crizotinib was purchased from Selleck Chemicals. G341-0312 was
purchased from ChemDiv company. They were dissolved in DMSO to a 50
mM or 20 mM concentration and stored in small aliquots at
-20.degree. C. until further use. Antibodies to GAPDH, ALK, p-ALK
(1282/1283), p-AKT (Ser473), p-ERK (Thr202/Thy204), ERK were
purchased from Cell signaling Technology.
H2228 (which express EML4-ALK fusion kinases, namely variant 3) and
BEAS-2B cells were obtained from the American Type Culture
Collection and cultured in environment of 5% CO.sub.2 at 37.degree.
C. in RPMI-1640 medium supplemented with 10% fetal bovine serum
(FBS), 100 units/mL penicillin, and 100 .mu.g/mL streptomycin.
Descriptive analytical data were presented as means SEM. Multiple
comparisons were evaluated by one-way analysis of variance (ANOVA)
followed by using Graph Prim5.0. Values of P<0.05 were
considered statistically significant.
Example 2A
Cytotoxic Effects of the Compound of Formula (Ic) Towards Cells
with ALK Chromosome Rearrangement
The cytotoxicity effect of the compound of Formula (Ic) on H2228
NSCLC cells (harboring ALK fusion) and human bronchial epithelial
(BEAS-2B) have been analyzed. H2228 cells were cultured in 96-well
plates at a density of 3.times.10.sup.3 cells/well, and were
cultured overnight for cell adhesion. Then the cells were treated
with DMSO or various concentrations of the compound of Formula (Ic)
for 72 h. To each well 10 .mu.L MTT (5 mg/mL) (Sigma) was added and
the cells were incubated for another 4 h at 37.degree. C., followed
by adding 100 .mu.L acidic isopropanol (10% SDS, and 0.01 mol/L
HCl). Finally, the optical density (OD) of each well was measured
at 570 nm by the Microplate Reader (Epoch, Winooski, USA). The cell
viability was calculated relative to untreated controls, with
results based on at least three independent experiments.
MTT assay showed that treatment with the compound of Formula (Ic)
was associated with a concentration-dependent significantly
decreased cell viability, with an IC.sub.50 value of 2.71.+-.0.92
.mu.M and lower cytotoxicity on normal lung cells BEAS-2B (see
FIGS. 6A and 6B and table 2).
TABLE-US-00002 TABLE 2 IC.sub.50 of the compound of Formula (Ic)
Cell line IC.sub.50 (.mu.M) H2228 2.71 .+-. 0.92 BEAS-2B 6.51 .+-.
1.08
Example 2B
Induction of Apoptosis in H2228 Cells by the Compound of Formula
(Ic)
H2228 cells (1.0.times.10.sup.5 cells/well) were allowed to attach
in a 6-well plate for 24 h. The cells were treated with various
concentrations of the compound of Formula (Ic), namely 1.25, 2.5
and 5 .mu.M, for 48 h. Subsequently, cells were trypsinized, washed
with PBS twice, then the cells were resuspended in a total volume
of 100 .mu.L binding buffer with 2 .mu.L Annexin-V FITC and 5 .mu.L
propidine iodide (PI). Finally, the cells were gently mixed and
incubated in the dark at room temperature for 15 min, before
further addition of 400 .mu.L of 1.times. Annexin-binding buffer,
the number of apoptotic cells was quantified using a Flow Cytometer
(BD Biosciences, San Jose, Calif., USA) within 1 h. Data were
analyzed by Flow Jo software.
Flow cytometry analysis showed that the compound of Formula (Ic)
induced cell death through induction of apoptosis of H2228 cells in
a concentration-dependent manner (FIG. 7A to FIG. 7F). Compared
with the control group, treatment on H2228 cells with the compound
of Formula (Ic) induced significant levels of cell apoptosis
especially in a concentration of at least 5 .mu.M (FIG. 7A to FIG.
7F).
Example 2C
Suppression of Colony Formation of H2228 Cells by the Compound of
Formula (Ic)
H2228 cells were seeded to six-well plate (1000/well). Then cells
were exposed to various doses of the compound of Formula (Ic),
namely 1.25, 2.5 and 5 .mu.M. After 14 days, colonies were fixed
with 4% paraformaldehyde for 15 min and stained with crystal violet
for 10-15 min. Finally, the staining solution was slowly washed off
with water and the cells were air dried. Clones with more than 50
cells were counted under a microscope.
The colony formation assay revealed that the compound of Formula
(Ic) inhibited the formation of H2228 cell colonies in a
dose-dependent manner (FIG. 8A to FIG. 8F). Especially when the
concentration of the compound of Formula (Ic) reached 5 .mu.M,
H2228 cells formed no visible colonies.
Example 2D
Suppression of ALK Phosphorylation and Anti-Apoptotic and Growth
Signaling Pathways Downstream to ALK by the Compound of Formula
(Ic)
Cells treated with different concentrations of the compound of
Formula (Ic) (1.25, 2.5 or 5 .mu.M) or 5 .mu.M of crizotinib or a
control group were washed twice with cold PBS then lysed in RIPA
lysis buffer containing protease and phosphatase inhibitors,
protein concentrations were determined by Bio-Rad protein Assay kit
(Bio-Rad, Philadelphia, Pa., USA). after equalizing the protein
concentrations of the samples, 5.times. laemmli buffer was added
and boiled at 100.degree. C. for 5 min. Equal amounts of the
protein samples (20-40 .mu.g per lane) were separated on a 10%
SDS-PAGE gel, then proteins were transferred onto Nitrocellulose
(NC) membrane at 300 mA for 2.5 hours at 4.degree. C., the
membranes was probed with primary antibodies overnight at 4.degree.
C. which was then exposed to 5% non-fat dry milk in TBST for 1 h at
room temperature with constant agitation and, then, rabbit or mouse
fluorescent antibodies (1:1000) were added to the membrane at room
temperature for 1 h. Visualization was performed using a LI-COR
Odessy scanner (Belfast, Me., USA). All primary antibodies were
diluted 1:1000, while their recommended secondary antibodies were
diluted 1:10000.
The anti-tumor efficacy of the compound of Formula (Ic) was
dose-dependent and led to significant suppression of ALK
phosphorylation as well as the downstream PI3K/AKT, MEK/ERK and
JAK/STAT3 signaling pathways (FIG. 9).
Example 2E
Binding Mode Between the Compound of Formula (Ic) and ALK
Kinase
Molecular docking calculation was performed to study the
interaction mode between the compound of Formula (Ic) and the
kinase binding domain of ALK by Induced Fit Docking module in
Schrodinger software (Schrodinger, Inc., New York, N.Y., 2009). The
studied compound of Formula (Ic) was prepared and optimized in the
LigPrep module. The 3D structure of ALK was derived from the PDB
database (PDB ID: 2XP2) and prepared using the Protein Preparation
Wizard. During the induced fit docking calculation, the
co-crystalized inhibitor crizotinib was used to define the active
site. The poses of the studied compound were evaluated by standard
precision (SP) docking score and the conformation with the highest
score is selected for binding mode analysis.
The binding affinity of the compound of Formula (Ic) and ALK was
evaluated by the SP docking score. The docking score of the
compound of Formula (Ic) is -9.413 Kcal/mol. The conformation of
the compound of Formula (Ic) has been superimposed with the
co-crystalized ligand crizotinib to compare their binding modes. As
shown in FIG. 10A, the scaffold of the compound of Formula (Ic)
overlapped well with the location of crizotinib, while both
molecules formed hydrogen bonds with residue Met1199 in the hinge
domain. As shown in FIG. 10B, the compound of Formula (Ic) was
buried in a hydrophobic pocket formed by Leu1122, Val1130, Leu1198,
Met1199, Ala1200, Gly1202, Leu1256 in the kinase binding domain
such as in the catalytic spine.
INDUSTRIAL APPLICABILITY
The present invention provides a new inhibitor, compound of Formula
(Ia), in particular of Formula (Ib) or (Ic), that can specific
target oncogenic ROS1 kinase and, furthermore, oncogenic ALK
kinase, which exhibits potent anti-cancer activity, especially in
NSCLC cell with ROS1 fusion gene or ALK fusion gene. The compound
of Formula (Ia) is suitable to suppress ROS1 and ALK
phosphorylation as well as respective downstream anti-apoptotic and
growth signaling effectors, including PI3K/AKT, MEK/ERK and
JAK/STAT3 signaling pathways. The present compound shows low
toxicity to normal lung epithelial cells, which can, thus, be used
as anti-cancer drug for targeting a subgroup of cancer patients who
harbor different forms of abnormalities in the ROS1 gene or ALK
gene, in particular chromosome rearrangements.
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